The regulation of protein synthesis in mammalian cells under stress conditions as well as the regulation of GCN4-specific translation in the yeast Saccharomyces cerevisiae under amino acid starvation conditions is mediated by phosphorylation of the eukaryotic translation initiation factor (eIF)-2. We have been studying how the mammalian PKR and HRI kinases and the yeast GCN2 kinase specifically recognize and phosphorylate serine-51 on the a subunit of eIF2 to regulate translation. One approach has been to study the vaccinia virus K3L protein, a pseudosubstrate inhibitor of PKR. We have established a system to suppress the toxicity of PKR expression in yeast by co-expressing K3L. Mutational analyses have revealed that residues near the carboxyl-terminus of K3L, that are conserved in eIF2a (residues 73-83), are critical for K3L activity. The corresponding residues in eIF2a are required for proper regulation of GCN4 expression suggesting that contacts over 20 residues from site of phosphorylation are important for kinase recognition of eIF2alpha. The suppression of PKR toxicity in yeast has also been used to identify a novel eIF2alpha kinase inhibitor from the baculovirus Autographa californica. The pk2 protein of baculovirus resembles a truncated eIF2 kinase domain and we have found that when expressed in yeast pk2 can inhibit both the PKR and GCN2 kinases. Similarly, the corresponding domain of GCN2 also acts as a dominant-negative inhibitor. In an analysis of substrate specificity of the eIF2 kinases it was found that all three eIF2 kinases readily phosphorylated threonine in place of eIF2alpha serine-51, and PKR was able to phosphorylate tyrosine at this position as well. In addition, PKR was found to autophosphorylate on tyrosine. These results identify PKR as a dual-specificity protein kinase. Finally, we have initiated a project examining a novel yeast protein, FUN12, which resembles a bacterial translation factor that performs the same role as eIF2.